Tremendous unmet clinical needs exist in musculoskeletal medicine. Novel strategies are required to safely promote bone formation in low turnover osteoporosis, osteoporosis in children with open epiphyses, avulsion injuries at sites of bone-tendon insertion, and fracture repair in the setting of underlying malignancy or vascular compromise. A fundamental understanding of the molecular mechanism governing osteoblast differentiation is essential for developing novel therapeutics to address these unmet needs. Notch signaling has emerged as an evolutionarily conserved cell-cell communication mechanism that controls cell fate in multicellular organisms. In the best-studied paradigms, upon ligand-induced cleavage by g-secretase, Notch receptors activate a transcription factor known as RBP-Jk in mammals, which in turn up-regulates expression of transcription repressors of the Hes/Hey family. It is the Hes/Hey family members that directly regulate the expression and/or function of cell-lineage specific transcription factors. Recently, by removing Notch receptors (Notch1 and 2) or core components of g-secretase (presenilin 1 and 2) in early limb mesenchyme, we discovered a physiological role for Notch signaling in osteoblast differentiation from progenitor cells. Specifically, loss of Notch signaling expands osteoblast numbers and augments trabecular bone mass in the appendicular skeleton. Importantly, specific members of the Hes/Hey family are reduced in Notch-deficient osteoblastic cells. Moreover, we found that Hes/Hey proteins physically associated with Runx2 and inhibited its activity. Thus, we hypothesize that 1) Notch acts through RBP-Jk to regulate Hes/Hey levels in osteoblast progenitors, and that 2) Hes/Hey proteins regulate osteoblast differentiation by modulating Runx2 activity. To test this hypothesis, we will pursue three specific aims to examine the potential bone phenotype in tissue- specific RBP-Jk knockout animals, and Hes/Hey mutant animals. We will also examine the role of the key molecules in osteoblast differentiation in vitro. Finally, we will begin to test the potential of inhibiting Notch signaling as a novel bone anabolic strategy.